Researchers are becoming increasingly aware that heterogeneity in the central nervous system (CNS) is encoded during embryonic development and retained during adulthood. One class of cells exhibiting substantial heterogeneity is spinal motor neurons, which innervate specific muscle targets based on cues received during development and are critically impaired in diseases such as Amyotrophic Lateral Sclerosis (ALS) and smooth muscle atrophy (SMA). While human pluripotent stem cell (hPSCs) differentiated to spinal motor neurons could provide an excellent resource for studying and/or treating these diseases, current differentiation methods are unable to recapitulate the developmental cues necessary to achieve defined positional identity. Furthermore, whereas diseases like ALS are non-cell-autonomous and involve complex cell- cell interactions, isolated differentiation of spinal motor neurons may not appropriately represent the disease progression and phenotype in a dish. As such, this proposal broadly seeks to differentiate hPSCs to spinal motor neurons possessing defined positional identity, both as an isolated population and within an organized multicellular tissue structure. Aim 1 of this proposal focuses on differentiation of hPSCs to neural progenitors possessing a defined position along the rostral/caudal spinal cord axis by combinatorial treatment with patterning factors such as Wnt3a, fibroblast growth factor 8, retinoic acid, and growth differentiation factor 11. Aim 2 will use a combination of micro-contact printing, surface chemistry, and recombinant protein engineering to create neural tissue structures of defined size and shape. Aim 3 will utilize two microfluidic devices to optimize spina motor neuron differentiation. The first microfluidic device will employ an active gradient generator that will identify the concentration of sonic hedgehog on a per cell basis necessary to achieve high yield motor neuron differentiation. The second microfluidic device will employ a passive gradient generator to pattern the engineered neural tissue structure towards the ventral portion of the neural tube. Successful completion up to the first half of Aim 3 will yield spinal motor neurons with defined positional identity that will have potential applications in regenerative therapy, while successful completion of the second half of Aim 3 will yield spinal motor neurons that reside in a ventral tissue structure that can be used in high throughput screening of therapeutics to treat ALS.